Isolation Of Cardiomyocytes Research Articles

Abstract 29: Isolation of Cardiomyocytes Undergoing Mitosis with Complete Cytokinesis

Adult cardiomyocytes (CMs) fail to exit the cell cycle and result in polyploidization and multi-nucleation. This extremely limited renewal potential is a key barrier for cardiac regeneration after heart injury. The use of traditional cell cycle assays such as Ki67 or purine analogue administration does not allow for isolation of viable CMs in late-M phase. We hypothesize that a molecular beacon (MB)-based method could be developed to isolate a highly pure population of hiPSC-derived CMs capable of completing mitosis and cytokinesis. We first differentiated human PSCs into cardiomyocytes using the small molecules CHIR and IWR and then applied MBs targeting CDC20 mRNAs, followed by fluorescence-activated cell sorting (FACS). Validation of cell cycle-specific and mitosis-associated gene expressions of cell fractions sorted from G2 and M-phase were performed by RT-qPCR prior to transcriptional profiling by single-cell RNA sequencing. Gene set enrichment analysis (GSEA) confirmed the observation of the gene ontology (GO) analysis. The ‘GO_cytokinesis’ and the ‘GO_cell separation after cytokinesis’ gene sets were highly enriched and predominantly upregulated in the CDC20 (+) (M-phase) population compared with the CDC20 (-) (G2-phase) population. Furthermore, using these data sets we expect to develop robust surface marker based flow cytometry protocols at a number and purity allowing for statistically significant genome-wide integrative transcriptome and epigenome analysis. In conclusion, the isolation of CMs using CDC20 MB and DNA content dye proved to enrich for cells that were confirmed by GSEA to be CMs in cytokinesis.

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac.

Background and PurposeMyocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R.Experimental ApproachHearts from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocyte isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery and myocardial injury during reperfusion (LDH release and infarct size) were evaluated. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca2+ transients.Key ResultsSelective activation of either PKA or Epac was found to trigger a positive inotropic effect, which was considerably enhanced when both pathways were simultaneously activated. Only combined activation of PKA and Epac induced marked cardioprotection against I/R injury. This was accompanied by PKCε activation and repressed by inhibitors of PKA, Epac or PKC.Conclusion and ImplicationsSimultaneous activation of both PKA and Epac induces an additive inotropic effect and confers optimal and marked cardioprotection against I/R injury. The latter effect is mediated by PKCε activation. This work has introduced a new therapeutic approach and targets to protect the heart against cardiac insults.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

In Vivo Cannulation Methods for Cardiomyocytes Isolation from Heart Disease Models.

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation–contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.

Abstract 218: Cardiac Side Population Cells Give Rise to Cardiomyocytes in the Adult Heart

Therapies that enhance cardiac regeneration have the potential to improve heart failure outcomes. One strategy to enhance cardiac regeneration is by activating endogenous cardiac progenitor cells. Cardiac side population cells (cSPCs) are proposed progenitor cells that reside in the heart; however, their putative progenitor cell properties are based on cell culture data and transplantation studies performed with isolated cSPCs. To determine the endogenous regenerative potential of cSPCs in vivo , we generated a mouse model that harbors an Abcg2-driven, tamoxifen-inducible Cre recombinase and crossed it to a GFP reporter mice. One month after tamoxifen treatment, we obtained efficient GFP-labeling of side population cells with 47.0 ± 11.05% of bone marrow side population cells and 75.8 ± 10.87% of cSPCs. Importantly, during a one-month chase period, we observed a three-fold increase in GFP-labeled cardiomyocytes when compared to a 1-week chase period. We quantified the extent of GFP-labeling of cardiomyocytes using adult cardiomyocyte isolation, where we measured 0.8% GFP labeled cardiomyocytes after a one-month chase period. We also observed labeling of many other cell types in the heart, such as endothelial cells, smooth muscle cells, and pericytes. We are currently testing to what extent cardiac injury enhances cSPC derived cardiomyocyte formation. Using our mouse model that efficiently labels side population cells in vivo , we demonstrated that cSPCs contribute cardiomyocytes in the adult heart in vivo .

Isolation of Cardiomyocytes and Cardiofibroblasts for Ex Vivo Analysis.

Heart failure (HF) is a common clinical endpoint to several underlying causes including aging, hypertension, stress, and cardiomyopathy. It is characterized by a significant decline in the cardiac output. Cardiomyocytes are terminally differentiated cells and therefore, apoptotic death due to beta adrenergic (β-AR) signaling contributes to high attrition rate of these cells. Past treatments of HF offer some survival benefit to patients (e.g., the beta blockers), but at the expense of blocking the compensatory beta-adrenergic signaling in surviving cells. One prerequisite for developing new therapeutics is to be able to grow cardiomyocytes ex vivo, and test their apoptotic response to drugs. Here we describe methods for isolation and culturing of neonatal and adult calcium tolerant cardiomyocytes. Similarly, cardiofibroblasts can also be isolated using the same protocol and subsequently, immortalized with SV40 T-Antigen for ex vivo studies.

The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts

Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts.Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (KATP) channel subunits are expressed in cultured fibroblasts. KATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential.We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells.UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of KATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts in human fetal hearts. Hypoxia further increased the number of human fetal and rat neonatal myofibroblasts. However, chronically administered UDCA reduced the number of myofibroblasts and prevented hypoxia-induced depolarisation.In conclusion, our results show that the protective effect of UDCA involves both the reduction of fibroblast differentiation into myofibroblasts, and hyperpolarisation of myofibroblasts, most likely through the stimulation of potassium channels, i.e. KATP channels. This could be important in validating UDCA as an antifibrotic and antiarrhythmic drug for treatment of failing hearts and fetal arrhythmia.

Non-genetic Purification of Ventricular Cardiomyocytes from Differentiating Embryonic Stem Cells through Molecular Beacons Targeting IRX-4.

SummaryIsolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ∼98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca2+ transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

RationaleCentral questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.Methods and ResultsIn order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.ConclusionHere, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

Abstract 17375: In Utero Particulate Matter Exposure Produces Heart Failure and Electrical Remodeling at Adulthood

Introduction: In utero exposure to particulate matter through perinatal development has been demonstrated to produce cardiac dysfunction during adulthood. It is unknown what effect exposure to air pollution during the in utero period alone has on cardiac dysfunction and electrical remodeling in adulthood. We tested the hypothesis that adult mice exposed to concentrated particulate matter in utero would demonstrate global cardiac dysfunction as well as cellular electrical remodeling at adulthood. Methods: Female FVB mice were exposed either to filtered air (FA) or particulate matter with diameter less than 2.5 μm (PM2.5) at a concentration of ~ 51.69 μg/m3 for 6 h/day, 7 days/wk (consistent with exposure in a large metropolitan city) beginning at plug formation throughout pregnancy. Cardiac function was assessed via ECHO in male offspring at 12 wks of age, followed by sacrifice and isolation of ventricular cardiomyocytes from both groups of mice for electrophysiological recordings. Results: ECHO identified increased LVESd (2.25 ± 0.20 FA, 2.61 ± 0.35 PM2.5, P=0.0001) and LVEDd (3.89 ± 0.03 FA, 3.99 ± 0.038 PM2.5, P=0.04) dimensions and reduced PWTs (1.40 ± 0.05 FA, 1.26 ± 0.04 PM2.5, P=0.04) in mice exposed in utero to PM2.5. Morphological alterations were associated with lower systolic function as indicated by reduced fractional shortening% (43.6 ± 2.1 FA, 33.2 ± 1.6 PM2.5, P=0.0009) in PM2.5 exposed mice compared to FA controls. Electrophysiological recordings revealed significant prolongation of the action potential at 90% repolarization (APD90) in PM2.5 exposed mice compared to FA. (FIGURE) Conclusions: In utero exposure to relevant levels of particulate matter results in dilated cardiomyopathy and electrical remodeling. Future studies are warranted to determine the causes of, and the exposure thresholds resulting in this adverse cardiac remodeling.

Abstract 6: Novel Tools for Generation, Purification, and Analysis of Pluripotent Stem Cell Derived Cardiomyocytes

Pure and well-characterized cardiac cells derived from human pluripotent stem cells (hPSCs) are of high interest for cardiovascular disease modeling, drug safety studies and development of cell replacement strategies. Although several protocols for cardiac differentiation of hPSCs have been developed, major limitations are clone-to-clone variations in differentiation efficacy as well as heterogeneity of generated cardiomyocyte populations. Therefore, we have developed novel tools for cardiomyocyte differentiation, magnetic cell sorting-based purification and cell analysis using new antibody-conjugates enabling for flow cytometry- and immunofluorescence-based identification of cardiomyocytes and subtypes. hPSCs were maintained under xeno-free conditions in our recently developed StemMACS iPS-Brew XF medium to keep pluripotency for more than >20 passages and enable for efficient cardiac differentiation. We chose a monolayer differentiation protocol based on the timely regulated activation and inhibition of Wnt signaling by small molecules. In order to identify antibodies suitable for cardiomyocyte enrichment or depletion of non-myocytes, we performed a surface marker screen with more than 400 antibodies between days 10-20 of differentiation. Besides identification of new surface markers, our screen confirmed expression of recently published markers like CD172a and CD106. In order to evaluate kinetics of their expression and correlation with intracellular markers of cardiomyocytes and subtypes, we performed a flow cytometry-based analysis of marker expression. Our data indicate a dynamic expression pattern for both CD172a and CD106, either completely or partially overlapping with intracellular cardiomyocyte marker expression. Based on these data we developed magnetic cell separation procedures for the isolation of cardiomyocytes. Magnetically enriched cardiomyocytes initiated contractions after replating and could be stably maintained in culture. Taken together, we have developed novel tools supporting the workflow for efficient generation of PSC-derived cardiomyocytes, magnetic purification and flow cytometry or immunofluorecence-based characterization of cardiomyocytes.

A simplified protocol for the isolation and culture of cardiomyocytes and progenitor cells from neonatal mouse ventricles

The neonatal heart is a very useful tool for the study of biochemical pathways and properties of cardiomyocytes and as it has the potential to regenerate for a brief period of time from birth; it is also useful to study cardiac regeneration. However, as the heart matures, this proficiency for regeneration is reduced. This regenerative potential may be influenced by the microenvironment of the heart in the early stages of postnatal development and therefore, cell cultures derived at this stage may contain functional cardiomyocytes and progenitor cells. The aim of this study was to identify key steps in the isolation and culture of such early stage—neonatal mouse hearts to allow maximum migration of cardiomyocytes from the explant and their maintenance as functional, long term cultures. Explant cultures of mouse ventricles preserved 3-dimensional structure and generated migrating layers of cardiomyocytes that expressed alpha sarcomeric actin which could be further sub-cultured by enzymatic dissociation. Western blotting demonstrated expression of c-KIT, GATA4, alpha sarcomeric actin and connexin43 proteins after 20 days of explant culture. ACTA1, GATA4, and CX43 continued to express in five weeks old explant cultures while the c-KIT protein was expressed up to two passages during sub-culture. Real time PCR and SQRT PCR also demonstrated gene expression of cardiomyocyte markers in long term cultures. Migrating cells from the explants assembled into contracting spheroids after subculture and expressed the c-KIT protein. Progenitor markers CD44, CD90, and extracellular proteins, periostin and vimentin demonstrated the preservation of cellular heterogeneity in such cultures. Supplementation with Hydrocortisone maintained a cardioprotective environment and reduced the non-myocyte population. This is an optimized and efficient method for the generation of neonatal heart cultures that is not labor intensive and does not require supplementation with cytokines.

In situ single photon confocal imaging of cardiomyocyte T-tubule system from Langendorff-perfused hearts.

Transverse tubules (T-tubules) are orderly invaginations of the sarcolemma in mammalian cardiomyocytes. The integrity of T-tubule architecture is critical for cardiac excitation–contraction coupling function. T-tubule remodeling is recognized as a key player in cardiac dysfunction. Early studies on T-tubule structure were based on electron microscopy, which uncovered important information about the T-tubule architecture. The advent of fluorescent membrane probes allowed the application of confocal microscopy to investigations of T-tubule structure. Studies have now been extended beyond single cardiomyocytes to examine the T-tubule network in intact hearts through in situ confocal imaging of Langendorff-perfused hearts. This technique has allowed visualization of T-tubule organization in their natural habitat, avoiding the damage induced by isolation of cardiomyocytes. Additionally, it is possible to obtain T-tubule images in different subepicardial regions in a single intact heart. We review how this state-of-the-art imaging technique has provided important mechanistic insights into maturation of T-tubules in developing hearts and defined the role of T-tubule remodeling in development and progression of heart failure.

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation and cryopreservation of neonatal rat cardiomyocytes.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Molecular beacon-based detection and isolation of working-type cardiomyocytes derived from human pluripotent stem cells

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide a potential source of cells to repair injured ventricular myocardium. CM differentiation cultures contain non-cardiac cells and CMs of both nodal and working subtypes. Direct application of such cultures in clinical studies could induce arrhythmias; thus, further purification of working-type CMs from heterogeneous cultures is desirable. Here, we designed 10 molecular beacons (MBs) targeting NPPA mRNA, a marker associated with working-type CMs and highly up-regulated during differentiation. We examined these MBs by solution assays and established their specificity using NPPA-overexpressing CHO cells as well as hPSC-CMs. We selected one MB for subsequent CM subtype isolation using fluorescence-activated cell sorting because the signal-to-background ratio was the highest for this MB in solution assays and a linear correlation was observed between MB signals and the CM purity in differentiation cultures. Compared with cells with low MB signals, cells positively selected based on MB signal had higher expression levels of genes associated with working-type CMs and lower expression levels of genes associated with nodal-type CMs. Therefore, the MB-based method is capable of separating working-type CMs from nodal-type CMs with high specificity and throughput, potentially providing working-type CMs for biomedical applications.

Adenoviral transduction of FRET-based biosensors for cAMP in primary adult mouse cardiomyocytes.

Genetically encoded biosensors that make use of fluorescence resonance energy transfer (FRET) are important tools for the study of compartmentalized cyclic nucleotide signaling in living cells. With the advent of germ line and tissue-specific transgenic technologies, the adult mouse represents a useful tool for the study of cardiovascular pathophysiology. The use of FRET-based genetically encoded biosensors coupled with this animal model represents a powerful combination for the study of cAMP signaling in live primary cardiomyocytes. In this chapter, we describe the steps required during the isolation, viral transduction, and culture of cardiomyocytes from an adult mouse to obtain reliable expression of genetically encoded FRET biosensors for the study of cAMP signaling in living cells.

Storage using BDM or Blebbistatin Preserves Functional Measures of Unloaded Cardiomyocytes

Intact isolated cardiomyocytes are used to investigate the molecular mechanisms of contraction and to screen new therapeutic compounds. Typically, these studies require either daily isolation or culture of cardiomyocytes, which either costs investigators time and animals or alters myocyte function and morphology. Recently, investigators reported preserving canine cardiomyocyte function using N-benzyl-p-toluene sulphonamide (BTS), a skeletal muscle myosin ATPase inhibitor. We investigated the preservation of rat cardiomyocyte function using the common cardiac cross-bridge inhibitors 2,3-Butanedione monoxime (BDM), a phosphatase, and Blebbistatin, a selective myosin ATPase. We isolated cardiomyocytes from 6 mo old female Sprague Dawley rats. The isolate was divided for use over multiple days and storage conditions. Functional measurements (calcium transients and sarcomere length (SL) twitches) were obtained in a Tyrodes solution at 25°C beginning on the day of isolation (Day 0). Cells used on Days 1-4 post isolation were stored in a HEPES buffered solution with 0.5mM Ca⊥(2+) at 4°C: (1) without additional cross-bridge inhibitors (control), (2) +10mM BDM, and (3) +3µM Blebbistatin. Control (inhibition-free) cells showed reduced SL (<1.7µm diastolic), membrane blebbing, and altered functional measurements (calcium transients and SL twitches) within 24 hours. Cells stored in BDM maintained function for 24 hours but exhibited reduced diastolic SL and significant blebbing at 48 hours. Cells stored in blebbistatin showed excellent maintenance of diastolic SL and cell membrane quality more than 4 days post isolation. Calcium transients did not change after 4 days of storage in blebbistatin, but SL shortening was attenuated after 24 hrs. UV light exposure deactivated blebbistatin inhibition and restored SL shortening back to Day 0 magnitudes. Storage of rodent cardiomyocytes may help investigators reduce, replace, or refine experiments by allowing extended use of cardiomyocytes from single isolations.

Isolation of contractile cardiomyocytes from human pluripotent stem-cell-derived cardiomyogenic cultures using a human NCX1-EGFP reporter.

The prospective isolation of defined contractile human pluripotent stem cell (hPSC)-derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as NKX2.5 or MYH6, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the SLC8A1 (NCX1) gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation, NCX1cp-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1(+) cardiac stromal cells or CD31/PECAM(+) endothelial cells. Flow-assisted cytometrically sorted EGFP(+) fractions of differentiated cultures were highly enriched in both early (NKX2.5 and TBX5) and late (CTNT/TNNI2, MYH6, MYH7, NPPA, and MYL2) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human SLC8A1(NCX1) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.